The present invention relates to a method of treating mammals suffering from conditions associated with excessive phospholipase A.sub.2 activity and/or production by administering to the mammal an amount and/or a type of a tetracycline that is not effectively antimicrobial but which effectively inhibits excessive phospholipase A.sub.2 activity and/or production. Excessive phospholipase A.sub.2 activity and/or production has been implicated in several disease conditions including; rheumatoid arthritis and other tissue destructive conditions, sepsis, septic shock, multisystem organ failure, pancreatitis, malaria, psoriasis and inflammatory bowel diseases. A composition useful in the treatment of mammals suffering from conditions associated with excessive phospholipase A.sub.2 activity and/or production is provided as well.
Phospholipase A.sub.2 (PLA.sub.2) is a ubiquitous lipolytic enzyme that has been implicated as a possible mediator of inflammation. Pruzanski et al., Immuno. Today 12: 143-146 (1991). Specifically, PLA.sub.2 hydrolyses the 2-acyl position of glycerophospholipids, liberating free-fatty acids, mainly arachidonic acid and lysophosphatides. Granstrom, Inflammation 8: S15-25 (1984, suppl 5), O'Flaherty, Lab. Invest. 47: 314-329 (1982). Subsequently, it is believed that arachidonic acid is converted into a variety of proinflammatory eicosanoids. Trang, Semm. Arthritis Rheum. 9: 153-190 (1980), Williams, Br. Med. Bull. 39: 239-242 (1983).
As indicated above, one of the suggested mechanisms of inflammation involves the activation of the arachidonic acid cascade which results in the liberation of a variety of proinflammatory eicosanoids. Van den Bosch, Biochem. Biophys. Acta 604: 191-246 (1980), Vadas et al., Lab. Invest. 55: 391-404 (1986). More recently, it has been suggested that PLA.sub.2 controls the first step in the liberation of arachidonic acid from phospholipids. Vadas et al., Lab. Invest. 55: 391-404 (1986). It has also been suggested that the inflammatory process may be conceptualized as a four-stage event: 1) exposure to the injurious agent, 2) synthesis and release of proximal mediators, 3) synthesis and secretion of PLA.sub.2 and 4) synthesis and release of distal effectors. Pruzanski et al., Immuno. Today 12: 143-146 (1991).
In stage 1 of this proposed scenario several factors participate in phagocytic and pinocytic activity, while others act as mediators of inflammation.
During stage 2, it is believed that a large number of proinflammatory mediators are synthesized and released in response to an injurious agent. Included in these mediators are complement, proteases, the contact activation system, toxic oxygen radicals, the interleukins (IL) (Mizel, Faseb J. 3: 2379-2388 (1989)); tumor necrosis factor (TNF) (Beutler et al., New Enql. J. Med. 316: 379-385 (1987)); interferons (INF) (Nathan et al., in Inflammation (Gallin et al. eds.) Raven Press 229-251 (1988)); platelet-activating factor (PAF) (Braquet et al., Immunol Today 8: 345-352 (1987)); eicosanoids (Marcus in Inflammation (Gallin et al. eds.) Raven Press 129-137 (1988)); and others (Wolpe et al., FASEB J. 3: 2565-2573 (1989)).
It is during stage 3 of this proposed scenario, that PLA.sub.2 appears to be synthesized and secreted. At least two forms of PLA.sub.2 have been found in cells. One form is associated with organelle membranes and plasmalemma and the other, a soluble form, is located in lysosomes and probably in cytosol. Hsueh et al., Nature 290: 710-713 (1981). The soluble form may be secreted from the cells into intravascular, interstitial or intraarticular compartments. IL-1 and TNF not only activate membrane-bound PLA.sub.2 but also induce the synthesis and extracellular release of soluble PLA.sub.2. Pruzanski et al., Immunol Today 12: 143-146 (1991).
It is believed that many disease states and conditions, which exhibit inflammation as part of the immunological process, are associated with elevated levels of PLA.sub.2. Several experimental models, both in vivo and in vitro, demonstrate this possible relationship between elevated PLA.sub.2 levels and inflammation. For example, glycogen-induced peritonitis in rabbits was found to be associated with high levels of soluble PLA.sub.2 in peritoneal exudate fluid. Franson et al., J. Lipid Res. 19: 18-23 (1978). A similar PLA.sub.2 was also found in the ascitic fluid of rodents after intraperitoneal injection of casein or zymosan. Gans et al., Agents Actions 27: 341-343 (1989). Experimental endotoxic shock is a recognized model of systemic inflammation. In rabbits challenged intravenously with Escherichia coli endotoxin, plasma PLA.sub.2 activity rose 11-fold and correlated with the fall in mean arterial blood pressure. Vadas et al., Can. J. Physiol. Pharmacol. 61: 561-566 (1983). PLA.sub.2 s purified from the venoms of snakes or bees also produce profound hypotension in various species. Marsh et al., Toxicon. 18: 427-435 (1980); Huaung et al., Eur. J. Pharmacol. 118: 139-146 (1985). These data show that bacterial endotoxins induce the intravascular release of PLA.sub.2 which in turn is related to cardiovascular collapse.
The administration of PLA.sub.2 can also induce significant lung injury. This is particularly important in bacterial peritonitis and septic shock which are often complicated by acute lung injury, manifested as the adult respiratory distress syndrome (ARDS). Intravenous infusion of PLA.sub.2 results in decreased compliance, impaired gas exchange, sequestration and infiltration of neutrophils in the pulmonary vascular bed and alveolar spaces. Morgan et al., Ann. Surg. 167: 329-335 (1968); Stommer et al., Klin. Wochenschr. 67: 171-176 (1989). Intratracheal instillation of PLA.sub.2 induces an intense inflammatory response in rabbit lung. Shaw et al., Am. J. Pathol. 91: 517-530 (1978). Therefore, both circulating and locally produced endogenous PLA.sub.2 may contribute to pulmonary inflammatory changes.
PLA.sub.2 is also vasoactive and proinflammatory when administered by other routes. Intradermal injection of PLA.sub.2 induces sustained hyperemia (Vadas et al., Nature 293: 583-585 (1981)); and an acute inflammatory infiltrate (Pruzanski et al., J. Invest. Dermatol. 86, 380-383 (1986)). Intra-articular administration of PLA.sub.2 in rats causes an acute synovitis after a single injection and synovial lining cell hyperplasia after repeat injections. Vadas et al., Am. J. Pathol. 134: 807-811 (1989). Several studies have also documented the induction of edema in mouse and rat footpads after injection of PLA.sub.2. Vishwanath et al., Inflammation 12: 549-561 (1988); Cirino et al., Eur J. Pharmacol. 166: 505-510 (1989). Moreover, extracellular PLA.sub.2 alters the function of phagocytes. Co-incubation of human neutrophils and monocytes with PLA.sub.2 from synovial fluid results in marked superoxide generation and lysosomal enzyme release, but decreased chemotactic responsiveness.
Numerous in vivo studies have also demonstrated the possible correlation between elevated PLA.sub.2 levels and inflammation. High levels of PLA.sub.2 activity have been found in synovial fluid from the inflamed joints of patients with rheumatoid arthritis (RA), psoriasis and osteoarthritis. Pruzanski et al., J. Rheumatol. 12: 211-216 (1985). High Levels of extracellular PLA.sub.2 activity are also present in patients with acute bacterial peritonitis. Vadas et al., in Cell Activation and Signal Initiation (Dennis et al., eds.) Alan R. Liss 311-316 (1989). Septic shock in humans is consistently associated with a marked rise in serum PLA.sub.2 activity. In retrospective and prospective studies of Gram-negative septic shock, all patients had elevated serum PLA.sub.2 levels during the acute hypotensive phase, which normalized during convalescence. Vadas et al., Crit. Care Med. 16: 1-7 (1988). In all patients, serum PLA.sub.2 levels correlated directly with the magnitude and duration of circulatory collapse. Furthermore, serum PLA.sub.2 was consistently elevated and the magnitude of the early increase in PLA.sub.2 was prognostic of the outcome. Serum PLA.sub.2 levels are also correlated with the increased risk of adult respiratory distress syndrome (ARDS) in patients with sepsis. Vadas, J. Lab. Clin. Med. 104: 873-881 (1984).
The exoantigens of the malaria parasite, Plasmodium falciparum, share common properties with endotoxin (Jakobsen, et al., Parasite Immunol. 10: 593-606 (1988)); and the syndrome caused by P. falciparum may resemble that of septic shock (Clark, Lancet ii: 75-77 (1978)). Serum PLA.sub.2 levels are elevated as much as 1100-fold before anti-malarial therapy.
Increased levels of PLA.sub.2 have also been found in patients suffering from osteoarthritis (OA). Pruzanski, et al., J. Rheumatol. 12: 211-216 (1985). Although OA is considered primarily a degenerative process, inflammatory episodes of varying duration are recognized as an integral part of this disease. Ehrlich, in Osteoarthritis. Diagnosis and Management (Moskowitz et al., eds.) W. B. Saunders Co. 199-209 (1984); Revell et al., Ann. Rheum. Dis. 47: 300-307 (1988). The mechanism of inflammation in OA has not been elucidated, but recently the role of inflammatory mediators has emerged as an important pathogenetic factor. Pelletier et al., J. Rheumatol. 16: (Suppl. 18) 19-27 (1989); Shinmei et al., J. Rheumatol. 16: (Suppl. 18) 32-34 (1989).
The diseases and conditions discussed above are not meant to be all encompassing. As the mechanisms of other disease processes are elucidated, PLA.sub.2 may be implicated as a possible mediator of the inflammatory response in those diseases as well. What is evident is the presence of elevated PLA.sub.2 levels in the inflammatory process in numerous serious diseases.
In spite of classical therapies for the treatment of the above diseases, the concomitant inflammation associated with excess levels of PLA.sub.2 in these diseases remains a problem. It is apparent therefore that a need exists for therapeutic agents that inhibit the excessive activity and/or production of PLA.sub.2, thereby controlling or eliminating its effect in various disease conditions. The present invention is intended to address this need. In particular, the present invention has discovered that the use of certain tetracyclines inhibits the excessive activity and/or production of phospholipase A.sub.2 (PLA.sub.2). In addition, the tetracyclines of the present invention can be combined with other classical therapeutic agents, such as anti-inflammatory agents or other medications which have been routinely used to treat the specific conditions discussed above. Other classical medications have not been known to function as inhibitors of excessive PLA.sub.2 activity and/or production. In contrast to these other conventional medications, the tetracyclines of the present invention have clearly demonstrated the inhibition of excessive PLA.sub.2 activity and/or production through the use of certain tetracyclines which inhibit the activity and/or production of PLA.sub.2.
Tetracyclines constitute a family of well known natural and synthetic broad spectrum antibiotics. The parent compound, tetracycline, exhibits the following general structure: ##STR1##
The numbering system of the ring nucleus is as follows: ##STR2##
Tetracycline as well as the 5-OH (Terramycin) and 7-Cl (Aureomycin) derivatives exist in nature, and are well known antibiotics. Natural tetracyclines may be modified without losing their antibiotic properties, although certain elements of the structure must be retained. The modifications that may and may not be made to the basic tetracycline structure have been reviewed by Mitscher in The Chemistry of Tetracyclines, Chapter 6, Marcel Dekker, Publishers, N.Y. (1978). According to Mitscher, the substituents at positions 5-9 of the tetracycline ring system may be modified without the complete loss of antibiotic properties. Changes to the basic ring system or replacement of the substituents at positions 1-4 and 10-12, however, generally lead to synthetic tetracyclines with substantially less or effectively no antimicrobial activity. For example, 4-dedimethylaminotetracycline is commonly considered to be a non-antimicrobial tetracycline.
The use of tetracycline antibiotics, while effective, may lead to undesirable side effects. For example, the long term administration of antibiotic tetracyclines may reduce or eliminate healthy flora, such as intestinal flora, and may lead to the production of antibiotic resistant organisms or the overgrowth of opportunistic yeast and fungi.
In addition to their antibiotic properties, tetracyclines have been described for a number of uses. For example, tetracyclines are also known to inhibit the activity of collagen destructive enzymes such as mammalian collagenase, gelatinase, macrophage elastase and bacterial collagenase. Golub et al., J. Periodont. Res. 20: 12-23 (1985); Golub et al., Crit. Revs. Oral Biol. Med. 2: 297-322 (1991).
Tetracyclines, administered at both antimicrobial levels and non-antimicrobial levels, have been known to play a role in reducing collagenase and other collagenolytic enzyme activity as well as collagen breakdown. U.S. Pat. Nos. 4,666,897; 4,704,383; 4,935,411; 4,935,412. In addition, tetracyclines have been known to inhibit wasting and protein degradation of mammalian skeletal muscle, U.S. Pat. No. 5,045,538. In addition, tetracyclines have been demonstrated to enhance bone formation in osteoporosis, U.S. Pat. No. 4,925,833. We have now discovered that tetracycline exhibits an anti-phospholipase A.sub.2 activity.
U.S. Pat. No. 4,704,383 to McNamara et al. discloses that tetracyclines having substantially no effective antimicrobial activity inhibit collagenolytic enzyme activity in rats. McNamara et al. also report that non-antimicrobial tetracyclines reduce bone resorption in organ culture. Earlier, U.S. Pat. No. 4,666,897 to Golub, et al. disclosed that tetracyclines in general, including commercially-available antimicrobial forms of the drug, inhibit excessive mammalian collagenolytic enzyme activity resulting in decreased connective tissue breakdown including that which occurs during bone resorption.
There have been a number of suggestions that tetracyclines, including non-antimicrobial tetracyclines, are effective in treating arthritis in rats. See, for example, Golub et al., "Tetracyclines (TCs) Inhibit Matrix Metalloproteinases (MMPs): In Vivo Effects In Arthritic And Diabetic Rats, And New In Vitro Studies," Matrix, Suppl. No 1: 315-316 (1992); Greenwald et al. "CMT, A Metalloproteinase Inhibitor, Prevents Bone Resorption In Adjuvant Arthritis." Arthritis Rheum. 34 (#9 suppl): S66 (abstract #A6), abstract presented at 55th Annual Meeting, Amer. College of Rheumatology, Boston Mass., Nov. 18, 1991; Breedveld, "Suppression Of Collagen And Adjuvant Arthritis By A Tetracycline," Northeastern Regional Meeting Of The Amer. Rheum. Assoc., Atlantic City, N.J. Oct. 23-24, 1987. For a related commentary regarding the effect of non-antimicrobial tetracyclines on bone loss see Sipos et al., "The Effect Of Collagenase Inhibitors On Alveolar Bone Loss Due To Periodontal Disease In Desalivated Rats," abstract presented at Matrix Metalloproteinase Conference, Destin, Fla., Sep. 11-15, 1989.
The effect of tetracyclines has not been firmly established for human patients with rheumatoid arthritis and various studies have indicated contrary results. Thus, Skinner et al., Arthritis and Rheumatism 14; 727-732 (1971), reported no significant benefit from tetracycline therapy for human sufferers of rheumatoid arthritis even though Greenwald et al., reported in J. Rheumatol. 14: 28-32 (1987) that the oral administration of a tetracycline (minocycline) to humans with severe rheumatoid arthritis decreased the collagenase activity in the joint tissues. More recently, however, Breedveld J. Rheumat. 17: 43 (1990) administered to humans, with rheumatoid arthritis, minocycline over a 16 week time period and reported a statistically significant improvement in a number of parameters of this disease, e.g. grip strength, erythrocyte sedimentation rate, etc. However, this study was not a placebo-controlled double blind study.
The use of tetracyclines in combination with non-steroidal anti-inflammatory agents has been studied in the treatment of inflammatory skin disorders caused by acne vulgaris. Wong et al., Journal of American Academy of Dermatology 11: 1076-1081 (1984), studied the combination of tetracycline and ibuprofen and reported that tetracycline was an effective agent against acne vulgaris, while ibuprofen was useful in reducing the resulting inflammation by inhibition of cyclooxygenase. Funt, Journal of the American Academy of Dermatology 13: 524-525 (1985), disclosed similar results by combining anti-microbial doses of minocycline and ibuprofen.
Based on the foregoing, tetracyclines, including their chemically modified analogs, have been found to be effective in different treatments. However, there has been no suggestion or indication that chemically modified tetracyclines inhibit excessive PLA.sub.2 activity and/or production.
The present invention is intended to provide a means for inhibiting the excessive PLA.sub.2 activity and/or production associated with many disease states. The present invention demonstrates that chemically modified analogs of tetracycline, which have lost their antimicrobial efficacy, have a novel new use, that is, the ability to inhibit excessive PLA.sub.2 activity and/or production. This non-antimicrobial property of tetracyclines reduces the severe inflammatory complications associated with excessive PLA.sub.2 activity and/or production present in many disease states.